CN101045721B - Preparation of porcelain vine element unsaturated sodium salt and its application - Google Patents

Abstract

This invention relates to a new ampeloptin unsaturation sodium salt and its recipe and application. This ampeloptin unsaturation sodium salt compare with ampeloptin, the physicochemical property occur notable change; by animal toxicity test proveing, both toxicity occur essentiality change; by pharmaco- experment justifing, ampeloptin unsaturation sodium salt possess synergy to many antineoplastic drug, can greatly reduce tumour drug using dosage, instead of impact curative effect.

Description

The preparation of porcelain vine element unsaturated sodium salt and application thereof

Technical field

The present invention relates to pharmaceutical field, more specifically, relate to the unsaturated salt of a kind of new ampelopsin, and method for making and purposes.

Background technology

Ampelopsin (AMP) is a kind of known compound from plant, and the molecular formula of its dihydrate is: C ₁₅H ₁₂O ₈2H ₂O, structural formula is as follows:

Ampelopsin (AMP) is proved to be has antineoplastic pharmacological action, and AMP and other tumour medicine compatibilities use simultaneously, can reduce the using dosage of tumour medicine significantly, thereby reaches the effect that reduces the tumour medicine side effect.

AMP also exists very big obstacle, and this mainly is because the solubleness of AMP in the aqueous solution is very low, needs to adopt Virahol, DMSO, organic reagent hydrotropies such as DMF.And organic reagent exists bigger toxicity, is not suitable as the use of clinical medicine.On the other hand, AMP itself also has bigger toxicity, and this also brings some problems for the security aspect of drug use.

Therefore, this area presses for the toxic method of exploitation attenuating ampelopsin.

Summary of the invention

Purpose of the present invention just provides the toxic method and formulation of a kind of remarkable attenuating ampelopsin.

In a first aspect of the present invention, a kind of ampelopsin salt or derivatives thereof is provided, described ampelopsin salt is to substitute the formed salt of hydrogen atom on the ampelopsin with monovalent cation, and describedly is replaced by unsaturated substituting.

In another preference, described derivative is hydrate or solvate (solvate).

In another preference, described ampelopsin salt has shown in the formula I:

C ₁₅H ₆O ₈H _αM _β(I)

In the formula,

M is the monovalent cation that is selected from down group: Li ⁺, K ⁺, Na ⁺, NH ₄ ⁺Or its combination;

α+β＝6，2≤β≤5。

In another preference, M is Na.

In another preference, described ampelopsin salt is dihydrate or the pentahydrate with porcelain vine element unsaturated sodium salt.

In another preference, described derivative is α=2, the hydrate that contains the unsaturated sodium salt ampelopsin of β=4, and molecular formula is: C ₁₅H ₈O ₈Na ₄5H ₂O.

In another preference, described pentahydrate has the following formula structure:

In a second aspect of the present invention, a kind of method for preparing ampelopsin salt or derivatives thereof of the present invention is provided, comprise step:

(a) with the reaction of the salt forming agent shown in ampelopsin and the formula II, form the ampelopsin salt shown in the formula I;

[0022]Ampelopsin+M _mZ → C ₁₅H ₆O ₈H _αM _β

[0023]Formula II formula I

In the formula,

M is the monovalent cation that is selected from down group: Li ⁺, K ⁺, Na ⁺, NH ₄ ⁺Or its combination,

Z is the negatively charged ion that is selected from down group: HCO ₃ ^-, CO ₃ ^2-, PO ₄ ^3-, HPO ₄ ^2-, H ₂PO ₄ ^-, Ac ^-, or its combination;

M is 1,2 or 3,

α+β＝6，2≤β≤5。

Wherein in the step (a), the ratio of the mole number of M is 1: 2 to 1: 5 in ampelopsin mole number and the salt forming agent;

(b) isolate ampelopsin salt or its hydrate of formation.

In another preference, described salt forming agent is selected from down group: sodium bicarbonate, yellow soda ash or its combination.

In another preference, be reflected in aqueous solvent or the water in the described step (a) and carry out, temperature is 4-80 ℃.

In a third aspect of the present invention, a kind of pharmaceutical composition is provided, it contains ampelopsin salt or derivatives thereof of the present invention and pharmacy acceptable salt.

In another preference, described pharmaceutical composition is injection, solution, tablet, lyophilisate or capsule.

In another preference, described pharmaceutical composition contains 0.2ug～500mg/ml ampelopsin salt.

In another preference, described pharmaceutical composition also contains extra antitumor drug.

In another preference, described antitumor drug is selected from down group: carboplatin, 5FU, Zorubicin, CTX, colchicine or its composition.

In a fourth aspect of the present invention, a kind of method for the treatment of tumour is provided, it comprises step: the ampelopsin salt or derivatives thereof of the present invention of giving the object significant quantity safe in utilization of needs treatment.

In another preference, also be included in use before the ampelopsin salt or derivatives thereof, afterwards or among, use extra antitumor drug.

In another preference, described safe and effective amount be use 1-5000mg ampelopsin salt/people/time.

In a fifth aspect of the present invention, a kind of method of pharmaceutical compositions is provided, it comprises step:

(a) the ampelopsin salt or derivatives thereof shown in the formula I is mixed with pharmaceutically acceptable carrier, thereby forms pharmaceutical composition:

C ₁₅H ₆O ₈H _αM _β (I)

In the formula,

M is the monovalent cation that is selected from down group: Li ⁺, K ⁺, Na ⁺, NH ₄ ⁺Or its combination;

α+β＝6，2≤β≤5。

In another preference, in step (a), also comprise and sneak into extra antitumor drug.

In another preference, described antitumor drug is selected from down group: carboplatin, 5FU, Zorubicin, CTX, colchicine or its composition.

In a sixth aspect of the present invention, the purposes of ampelopsin salt or derivatives thereof of the present invention is provided, they are used to prepare anti-tumor drug, perhaps use with other tumour medicine compatibilities, thereby reduce other tumour medicine side effects.

Description of drawings

Fig. 1 has shown that AMP-Na and AMP-DMSO are in 48h and the 72h comparison to the lethal effect amount effect curve of K562 cell.

Embodiment

The inventor is surprised to find that through extensive and deep research, uses the salt of weak acid of monovalent base metal ion that AMP is carried out the salify modification, and carries out unsaturated substituting for last 6 the free hydrogen atoms of AMP, can significantly change its physico-chemical property.Adopt saturatedly to substitute or do not do anyly when alternative, the dissolving properties of AMP is difficult for dissolving or progressively folds into crystallite substantially without any change in the aqueous solution, cause injecting the local excitation reaction of back to human body.And adopt monovalent cation to carry out unsaturated substitute (as substituting than 1 mole of AMP) with 2-5 mole monovalent cation, just can significantly improve the solubleness of AMP.

Term

As used herein, term " AMP " refers to ampelopsin.

As used herein, term " AMP-M " refers to modify method with salify, ampelopsin is carried out the part salify modify, and the unsaturated monovalent cation ampelopsin salt that obtains (is called for short down: AMP-M).

As used herein, term " ampelopsin salt or derivatives thereof " refers to ampelopsin salt, its hydrate or solvate.

As used herein, term " ampelopsin salt of the present invention " or " undersaturated ampelopsin salt ", " the unsaturated ampelopsin salt of monovalent cation " are used interchangeably, and all the finger divide to replace the hydrogen in 6 hydroxyls in the ampelopsin and the salt that forms.In addition, go back the reactive derivative (as hydrate or solvate) that term also can comprise ampelopsin salt.

As used herein, term " AMP-Na ₄" refer to replace in the ampelopsin molecule 4 hydrogen in 6 hydroxyls and the salt or the derivative (as hydrate) that form with 4 sodium ions.

Salt forming agent

Can be used for salt forming agent of the present invention and be not particularly limited, can be conventional strong base weak acid type salt.Can be the strong base weak acid type salt that forms by strong base ion and weak acid root.

Representational weak acid root comprises Ac, HPO ₄, H ₂PO ₄, HCO ₃, CO ₃Deng, preferably adopt HCO ₃, CO ₃, more preferably adopt CO ₃, make the pH value more stably reach neutral range.

Representational strong base ion is Li ⁺, K ⁺, Na ⁺, NH ₄ ⁺Or its combination; It more preferably is sodium ion.

In another preference, described salt forming agent is selected from down group: sodium bicarbonate, yellow soda ash or its combination.

The preparation method

The present invention also provides a kind of method for preparing ampelopsin salt, and it comprises step:

(a) with the reaction of the salt forming agent shown in ampelopsin and the formula II, form the ampelopsin salt shown in the formula I;

[0066]Ampelopsin+M _mZ → C ₁₅H ₆O ₈H _αM _β

[0067]Formula II formula I

In the formula,

M is the monovalent cation that is selected from down group: Li ⁺, K ⁺, Na ⁺, NH ₄ ⁺Or its combination,

Z is the negatively charged ion that is selected from down group: HCO ₃ ^-, CO ₃ ^2-, PO ₄ ^3-, HPO ₄ ^2-, H ₂PO ₄ ^-, Ac ^-, or its combination;

M is 1,2 or 3,

α+β＝6，2≤β≤5。

Wherein in the step (a), the ratio of the mole number of M is 1: 2 to 1: 5 in ampelopsin mole number and the salt forming agent;

(b) isolate ampelopsin salt or its hydrate of formation.

Usually, be reflected in the described step (a) in aqueous solvent (with water and ethanol mixed solvent) or the water and carry out, temperature is 4-80 ℃.

Pharmaceutical composition

Ampelopsin salt of the present invention can be used for treatment or adjuvant therapy of tumors.Usually, ampelopsin salt of the present invention can be formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can change to some extent with being prepared Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.

Ampelopsin salt of the present invention can be directly used in disease treatment, for example, is used for the treatment of tumour aspect.When using ampelopsin salt of the present invention, also can use the other treatment agent simultaneously, as carboplatin, 5FU, Zorubicin, CTX, colchicine or its composition etc.

The present invention also provides a kind of pharmaceutical composition, and it contains ampelopsin salt of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount (as 0.001-99.9wt%, more preferably 0.01-90wt%).

This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.

A kind of preferred drug substances is that the ampelopsin salt concn is the pure solution of 0.2ug～500mg/ml.

In addition, in pharmaceutical composition of the present invention, can add or not add other chemical reagent of no pharmacological action as the composite composition of PH conditioning agent, stablizer, solubility promoter or the like.

Pharmaceutical composition of the present invention can be made into unit dosage form or polynary formulation, can separately or mix and use, and can be applied to treat the medicament and/or the medicament synergistic agent of tumour.

When making pharmaceutical composition, be that the ZZ albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

Major advantage of the present invention is:

(a) solubleness height, good stability.Phenomenons such as adopt the AMP-M after salify is modified, dissolving properties improves greatly, and this dissolving properties is very stable, and continuous 15 days stability observing do not see any muddiness, separate out; And the AMP-M behind this salify can not influence quality product or change its physico-chemical property with widely used clinically phosphoric acid buffer arbitrary proportion dilution allotment at present.

(b) toxicity reduces.Modified AMP-M of the present invention, give mouse large vol injection after, not seeing has any toxic side effect, adopt maxima solubility and maximum capacity dispenser after, the animal mld is greater than 2g/kg; And use the dosage of AMP at 1g/kg, the half animal causes death.Therefore, the toxicity character of visible AMP-M changes greatly; Be more suitable for the judge of medicine aspect security.

(c) can use with other tumour medicine compatibilities.Modified AMP-M can use with the kinds of tumors compatibility of drugs, can reduce drug use dosage significantly, reduces drug toxicity, gives full play of the effect of tumour medicine, is a kind of intensive synergistic agent.Simultaneously, owing to changed dissolving properties and toxicity character, make heavy dose of AMP-M that directly uses become possibility, therefore, AMP also can be applied to clinical as a kind of anti-tumor drug preparation separately.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise umber and percentages calculate.

The preparation of embodiment 1AMP-Na salt and solubleness and stability are measured

AMP and NaHCO ₃Calculate consumption by different molar concentration rates (promptly 1: 1,1: 2,1: 3,1: 4), AMP is used 5% dissolve with ethanol earlier, press concentration ratio again and add different amount NaHCO ₃Reach distilled water and make it abundant dissolving, observe dissolution time, steady dissolution and also measure the pH value of solution value.Get AMP and Na ₂CO ₃, press mass ratio AMP: Na ₂CO ₃(w/w)=also preparing AMP-Na respectively is subjected to test solution for 5: 2,5: 3,5: 4 calculating medicines, distilled water consumptions, get a certain amount of test solution that is subjected to respectively with physiological saline or pH=6,6.5,7.0,7.4,0 times of dilution of 8.0 phosphate buffer 1s, observe the stability of placement solution under pH value, dissolving situation and the different condition of solution.

The result:

AMP and NaHCO ₃Salify dissolving situation sees the following form:

* room temperature is 24 ℃.

AMP and Na ₂CO ₃Salify dissolving situation:

Press AMP: Na ₂CO ₃(w/w)=and preparation in 5: 2, AMP can not dissolve fully, has molecule to separate out.

Press AMP: Na ₂CO ₃(w/w)=and preparation in 5: 3, AMP can dissolve fully, and concrete outcome sees the following form:

* AMP-Na is prepared by test solution: AMP 0.025g+Na ₂CO ₃0.015g+ distilled water 1ml

* room temperature is 24 ℃.

Conclusion:

The pure product of AMP are white powder, and are water insoluble, are dissolved in dimethyl sulfoxide (DMSO).And with AMP respectively with NaHCO ₃Or Na ₂CO ₃Make the AMP sodium salt solution by a certain percentage, the solvability of AMP is significantly improved.

By comparing AMP and NaHCO ₃And Na ₂CO ₃Salify solvability and stability can be with AMP and Na ₂CO ₃By mole number Na: AMP=4 in the solution: 1 prepares, and this moment, AMP can dissolve immediately fully, and stability is reliable, and solution is after pH=6,6.5,7.0,7.4,0 times of dilution of 8.0 phosphate buffer 1s, and 4 ℃ to preserve solvabilities constant.

Embodiment 2AMP-Na ₄The pharmacological action of salt and with the comparison of AMP

The utilization mtt assay, relatively whether AMP-Na and the external lethal effect to human leukemia K562 cell of AMP-DMSO there are differences aspect the pharmacodynamic action to observe AMP sodium salt preparation and traditional DMSO dissolving method.Through relatively AMP-Na and AMP-DMSO at 48h and 72h lethal effect to the K562 cell, the IC50 value that found that AMP-DMSO is 32.27 μ g/ml, the IC50 value of AMP-Na is 29.56 μ g/ml, and the two is to human leukemia K562 cells in vitro lethal effect no significant difference.

Medicine adopts: AMP powder particle 0.5g/ props up (available from chemistry teaching and research room of Zhongshan Medical Univ.), Na ₂CO ₃(analytical pure), distilled water, phosphate buffered saline buffer (PH=6.5,7.4), methyl-sulphoxide (DMSO), 5-fluor-uracil (5-FU, Shanghai Xudong Hipu Medicine Co., Ltd produces, lot number: H31020593), the RPMI1640 nutrient solution, calf serum, MTT, 10%SDS.

Preparation as follows:

1. AMP-Na:, select AMP: Na according to the method for embodiment 1 ₂CO ₃(w/w)=5: 3 ratio prepares AMP-Na solution, uses the pH=6.5 phosphate buffered saline buffer with its 10 times of dilutions again, makes the AMP-Na stock solution.With the pH=7.4 phosphate buffered saline buffer AMP-Na stock solution is made into cell concentration.

2. AMP-DMSO:AMP is with 5%DMSO dissolving and be made into cell concentration.

3. 5-FU: 5-FU is made into cell concentration with physiological saline.

More than each medicine be and face with preceding fresh preparation, drug cell is 100,50,25,12.5,6.25 μ g/ml with final concentration.

Tumor cell line:

Human leukemia K562 cell is available from Shanghai cell biological institute of Chinese Academy of Sciences cell bank.

Cell cultures:

Used cell is cultivated for the RPMI RPMI-1640, contains penicillin, the Streptomycin sulphate of 10% calf serum and 100U/ml in the nutrient solution.

The experiment grouping:

Blank group (solvent+RPMI1640 nutrient solution), negative control group (solvent+cell suspension), positive controls (5-FU), DMSO+AMP group and AMP-Na group are established in experiment.

Experimental technique:

1. collect the K562 cell that is in logarithmic phase, 1000 rev/mins, centrifugal 5 minutes, abandon supernatant, adjust cell concn to 1 * 10 with the RPMI RPMI-1640 ⁵/ ml.

2. add the different concns medicine successively with the 10ul/ hole in 96 well culture plates, add cell suspension or nutrient solution, put CO with the 90ul/ hole ₂Incubator is cultivated.

3. 4h adds MTT with the 10ul/ hole before experiment stops, and continues to be cultured to 48 or 72 hours, adds 10%SDS with the 100ul/ hole again and stops experiment.

4. culture plate is measured light absorption value with micro-oscillator concussion 10 minutes in wavelength 570nm place, prints and the record result.

Data statistics:

Be calculated as follows inhibiting rate:

Inhibiting rate (%)=[(negative OD average-blank OD average)-(being subjected to the blank OD average of reagent OD average-be subjected to reagent)]/(negative OD average-blank OD average) * 100/%

By drug level inhibiting rate is carried out linear regression, calculate the IC50 value.

Experimental result:

By measuring amount effect curve, relatively AMP-Na and AMP-DMSO are in 48h and the 72h power to the lethal effect of K562 cell, the IC50 value of AMP-DMSO and AMP-Na is respectively 32.27 μ g/ml and 29.56 μ g/ml when found that cell cultures 48h, the IC50 value of AMP-DMSO and AMP-Na is respectively 4.09 μ g/ml and 4.23 μ g/ml during cell cultures 72h, and the two is to human leukemia K562 cells in vitro lethal effect no significant difference (Fig. 1).

The IC50 of AMP-DMSO and AMP-Na relatively

Conclusion:

The AMP dimethyl sulfoxide (DMSO) was the solvent hydrotropy in the past, but this method solubleness is low, and this solvent is difficult to be applied to clinical; And when testing in carrying out external, body, there is certain interference in dimethyl sulfoxide (DMSO) to experimental result.The AMP-Na solution of the method preparation that the present invention addresses, and regulate its pH to 7.4 with the pH=6.5 phosphate buffered saline buffer, whether investigate AMP sodium salt preparation and DMSO hydrotropy method there are differences aspect pharmacodynamic action, this experiment utilization mtt assay, compared AMP-Na and AMP-DMSO 48h and 72h to human leukemia K562 cells in vitro lethal effect, found that AMP-Na is similar to the effect of AMP-DMSO to K562 cells in vitro lethal effect.And AMP-Na is water-soluble, can be used for clinically, is the optimal formulation method of AMP.

Embodiment 3AMP-Na ₄The antitumor action of salt and synergism

Be the antitumor action of research AMP-Na, and determine its independent medication to the influence of tumor growth and with the synergy of chemotherapeutic combined utilization, in the present embodiment, tumor-inhibiting action in the body of AMP-Na is tested with the KM mouse of lotus S180 sarcoma.Experiment observed altogether the independent medication of AMP-Na and with the synergy of 3 kinds of chemotherapeutic (CTX, 5-FU and carboplatin) combined utilization.Mouse armpit subcutaneous vaccination mouse S180 sarcoma cell 0.5-1 * 10 ⁶Cell/only, inoculate the administration of dividing into groups back next day.Drug combination group elder generation abdominal injection chemotherapeutic, abdominal injection AMP-Na again after 15 minutes, administration is 1 time next day that the two being, and administration is 6-8 time altogether.Get the knurl piece during experiment terminal point, claim knurl heavy, calculate tumour inhibiting rate.Also learning processing P＜0.05 by statistics with tumour inhibiting rate 40% serves as effectively, observes the tumor killing effect that is subjected to reagent.Experimental result finds that the independent medication of AMP-Na do not have or more weak antitumor action is only arranged, and AMP-Na and chemotherapeutic are share the S180 sarcoma is had the obvious synergistic antitumor action.Concrete experimental result is as follows:

Prepare AMP-Na by the method among the embodiment 1, the phosphate buffer 1 80ml with PH=6.5 adjusts the solution pH value again, makes PH=7.2.Solution through 0.45, the degerming of 0.22um membrane filtration, packing, 4 ℃ of preservations are standby.AMP-Na faces with before using administration after the phosphate buffered saline buffer of autoclaved PH=7.4 is mixed with purpose concentration.

1.25-FU, CTX and carboplatin be with preceding with the fresh preparation of physiological saline.

Animal is adopted Kunming mouse, the SPF level, and 18-22g, the male and female dual-purpose is available from Lanzhou University's Experimental Animal Center (production licence number: the moving word 14-005 of doctor).Use other animal of identity with a collection of experiment.

Tumor cell line is the strain of mouse S180 sarcoma cell, all goes down to posterity to protect with the Kunming mouse intraperitoneal and plants.

Transplanted tumor plantation and pharmacological agent: aseptic S180 and the H22 cell suspension (2.5 * 10 got ⁶Individual cell/ml), every mouse 0.2ml are inoculated in that the right side of mice armpit is subcutaneous (to be equivalent to every mouse inoculation 5 * 10 ⁵Individual cell).Inoculation random packet next day gives pharmacological agent.Medication is AMP-Na 50,75,112,167mg/kg, 0.1ml/10g, ip; During drug combination, give after the chemotherapeutic 15 minutes and give AMP-Na again.Be administered once 6-8 time altogether next day that both being equal.

The calculating of tumor weight mensuration and tumour inhibiting rate (%): weigh next day after the last administration, puts to death animal, dissects and strip the knurl piece, claims knurl heavy.Be calculated as follows tumour inhibiting rate (%):

Tumour inhibiting rate (inhibition rate of tumor growth, %)=heavy (g) * 100% of (the average knurl of the negative control group average knurl of heavy (g)-administration group heavy (g))/average knurl of negative control group

Effectively criterion: tumour inhibiting rate (being inhibition rate of tumor growth)＜40% is for invalid; Tumour inhibiting rate 40% is also learned by statistics and handled P＜0.05 is effective.

Experimental result:

1.AMP-Na with the synergy of CTX when treating murine sarcoma S180.

Compare with positive controls, AMP-Na and CTX drug combination group can make tumor weight obviously alleviate, and there was a significant difference; Wherein the knurl of AMP-Na 50,75mg/kg and CTX drug combination group heavily is starkly lower than the independent medication group of CTX.The tumour inhibiting rate of AMP-Na 50,75,112, the independent medication group of 167mg/kg is respectively 7.66,6.84 ,-3.86 and-16.25%; The tumour inhibiting rate of AMP-Na 50,75,112,167mg/kg and CTX drug combination group is respectively 64.45,68.07,46.13 and 64.52%; CTX group tumour inhibiting rate is 53.30%.

AMP-Na and the CTX synergy when treatment murine sarcoma S180

* represent p＜0.05, * * represents p＜0.01

2.AMP-Na with the synergy of 5-FU when treating murine sarcoma S180.

Compare with positive controls, AMP-Na and 5-FU drug combination group can make tumor weight obviously alleviate, and there was a significant difference, and the inhibiting rate of tumour is had the dose-dependently relation; Wherein the knurl of AMP-Na 50,75,112mg/kg and 5-FU drug combination group heavily is starkly lower than the independent medication group of 5-FU.The tumour inhibiting rate of AMP-Na 50,75,112, the independent medication group of 167mg/kg is respectively 4.43,31.28,33.49 and 4.45%; The tumour inhibiting rate of AMP-Na 50,75,112,167mg/kg and 5-FU drug combination group is respectively 57.75,51.87,59.21 and 36.65%; 5-FU group tumour inhibiting rate is 45.55%.

AMP-Na and the 5-FU synergy when treatment murine sarcoma S180

* represent p＜0.05, * * represents p＜0.01

3.AMP-Na with the synergy of carboplatin when treating murine sarcoma S180.

Compare with positive controls, AMP-Na and carboplatin drug combination group can make tumor weight obviously alleviate, and there was a significant difference; Wherein the knurl of AMP-Na 50,75,112,167mg/kg and carboplatin drug combination group heavily is starkly lower than the independent medication group of carboplatin.The tumour inhibiting rate of AMP-Na 50,75,112, the independent medication group of 167mg/kg is respectively 23.22,3.99,13.87 and-7.47%; The tumour inhibiting rate that AMP-Na 50,75,112,167mg/kg and carboplatin are united with the medication group is respectively 65.11,54.35,66.37 and 59.26%; The tumour inhibiting rate of carboplatin is 49.52%.

AMP-Na and the carboplatin synergy when treatment murine sarcoma S180

* represent p＜0.05, * * represents p＜0.01

Conclusion

AMP-Na abdominal injection and 3 kinds of chemotherapeutic CTX, 5-FU and carboplatin combined utilization all have the effect of collaborative anti-mouse S180 sarcoma.AMP-Na 50-167mg/kg and chemotherapeutic 5-FU, CTX and carboplatin combined utilization can suppress the growth of mouse S180 sarcoma in various degree, compare with the carboplatin group with independent treatment with chemotherapy medicine 5-FU, CTX, and there was a significant difference.Dosage is that the effect of share anti-mouse S180 sarcoma of AMP-Na in the 75-112mg/kg scope and chemotherapeutic is more obvious.

Embodiment 4AMP-Na ₄The toxicity research of salt

For the acute toxicity effect of research AMP-Na, determine its LD50 value and maximum tolerated dose, and contrast with AMP to mouse.

Give AMP-Na intravenous injection with Kunming mouse, observe after the administration animal dead performance and dead distribution situation in 7 days.Chronic dead animal is dissected and got main organs and the visible pathology internal organs of naked eyes, and is fixing, does pathology section examination.As a result, the AMP-Na intravenous injection, the LD50 value of male and female mice is all greater than 2000mg/kg, LD0＞1000mg/kg.And the LD50 value of animal is 1000mg/kg in the AMP-DMSO group, shows and can significantly reduce toxicity.

Get AMP-PEG solution 5ml, add Na ₂CO ₃1.25g make AMP: N a ₂CO ₃(w/w)=5: 3, add physiological saline 36.25ml and 37%HCl 0.75ml, getting AMP-Na concentration is that (wherein the concentration of solvent is PEG 4000.03g/ml to 50mg/ml, propylene glycol 0.0445g/ml, ethanol 0.015g/ml), the pH value of final AMP-Na solution is 7.3.The AMP-Na stock solution is made into desired concn with physiological saline, presses 0.2ml/10g, abdominal cavity or intravenous administration.

Laboratory animal is a Kunming mouse, the SPF level, and 18-22g, the male and female dual-purpose is available from Lanzhou University's Experimental Animal Center (production licence number: the moving word 14-005 of doctor).AMP-Na solvent group and AMP-Na series dosage group are established in experiment respectively, and the result is seen in concrete grouping.Each is organized data and is enumeration data, carries out computational analysis with DAS software.

The result:

Intravenous administration result statistics of AMP-Na 1000mg/kg:

AMP-Na total amount 2000mg/kg divides intravenous administration result statistics 2 times:

Intravenous administration result statistics of AMP-DMSO total amount 1000mg/kg:

Conclusion:

Mouse LD50 value is greater than 2000mg/kg.Wherein buck to the maximum tolerated dose of AMP-Na greater than 2000mg/kg; Quiet notes LD ₀＞1000mg/kg; And when adopting DMSO to be solvent, LD50 is 1000mg/kg.

The chemical structure analysis of embodiment 5 ampelopsin sodium salts

To the AMP sodium salt of preparation among the embodiment 1, molecular formula is: C ₁₅H ₈O ₈Na ₄5H ₂O has carried out chemical structure analysis

[thermogravimetric analysis]

Room temperature (20 ℃) is to 3.41%, 80.2 ℃ to 176.0 ℃ weight loss 12.31% of 80.2 ℃ of weight losses, amounts to 15.72%, and the calculated value 16.57% of 4.5 crystal water conforms to substantially in this value and the sample.Be also shown in 119.6 ℃ in the dsc detected result and endotherm(ic)peak occurs, proved the existence of crystal water in this structure indirectly.

[ultimate analysis]

The result proves that the carbon in the sample, protium content measured value are respectively 35.87%, 3.66%, with calculated value (carbon, hydrogen are respectively 36.16%, 3.64%) basically identical.

[ion chromatography]

Sodium element content measured value in the working sample is 18.6%, conforms to substantially with calculated value (18.46%).

[ultra-violet absorption spectrum]

Ultra-violet absorption spectrum in water solvent shows that there is the fine structure of aromatic ring in the E band, 4 absorption peaks occur at 204.6nm, 206.1nm, 210.1nm and 218.1nm respectively; B band 1 absorption peak occurs at 328.3nm, than the absorption peak 292.2nm red shift of the ampelopsin (in methyl alcohol) of no sodium 36.1nm.

[nuclear magnetic resonance spectrum]

Instrument: U.S. Varian ^UNITYINOVA 500 superconduction pulse fourier transform nuclear magnetic resonance spectrometers

(1) hydrogen spectrum

¹H nuclear magnetic resonance spectrum data and parsing

Solvent: DMSO

Spectrum peak sequence number

Ampelopsin chemical shift δ (ppm)

Ampelopsin sodium salt chemical shift δ (ppm)

The ampelopsin sodium salt is through being neutralized to the chemical shift δ (ppm) of pH6

Split the branch peak number

Proton number

Remarks

a?

4.45?

4.05?

4.41?

d?

1?

3 CH

b?

4.94?

4.49?

4.90?

d?

1?

2 CH

c?

5.73?

?

5.73?

br?s?

1?

3 OH

d?

5.90?

5.05?

5.86?

d?

1?

8 CH

e?

5.94?

5.07?

5.90?

d?

1?

6 CH

f?

6.45?

6.28?

6.44?

s?

2?

2 '/6 ' CH

g?

8.16?

8.29?

8.30?

br?s?

1?

4 ' OH

h?

8.89?

?

8.88?

br?s?

2?

3 '/5 ' OH

i?

10.82?

?

10.90?

br?s?

1?

7 OH

j?

11.87?

12.3(br s)

11.88?

s?

1?

5 OH

[0192](2) carbon spectrum

¹³C nuclear magnetic resonance spectrum data and parsing

Spectrum peak sequence number	Ampelopsin chemical shift δ (ppm) (solvent DMSO)	Ampelopsin sodium salt chemical shift δ (ppm) (solvent D 2O)?	Multiplicity (DEPT)	Carbonatoms	Remarks
						A?	71.83?	71.64?	d?	1?	3 CH
B?	83.40?	83.72?	d?	1?	2 CH
						C?	95.16?	97.92?	d?	1?	8 CH
D?	96.20?	99.05?	d?	1?	6 CH
						E?	100.65?	99.82?	s?	1?	10 C
F?	107.19?	107.50?	d?	2?	2 '/6 ' CH
						G?	127.37?	126.34?	s?	1?	1 ' C
H?	133.63?	137.29?	s?	1?	4 ' C
						I?	145.86?	147.61?	s?	2?	3 '/5 ' C
J?	162.66?	161.72?	s?	1?	9 C
						K?	163.49?	162.84?	s?	1?	5 C
L?	166.95?	163.46?	s?	1?	7 C
						M?	197.58?	194.04?	s?	1?	C＝O?

Annotate: because the solubleness of ampelopsin sodium salt in the DMSO solvent is relatively poor, so adopt D ₂O makes solvent.

Can obtain following information from hydrogen spectrum and carbon spectrum data:

(1) the ampelopsin sodium salt is positioned at 12.3 (1H, br s) ppm place, has shown that 5 hydroxyl protons and 4 oxygen form intramolecular hydrogen bond.

(2) the hydrogen spectrum with ampelopsin compares, and ampelopsin sodium salt hydrogen spectrum disappears at the absorption peak of 3 OH, 7 OH, 3 '/5 ' OH.

(3) with the carbon of ampelopsin spectrum relatively, ampelopsin sodium salt carbon spectrum in the about displacement of absorption peak of 8 CH, 6 CH, 4 ' C, 3 '/5 ' C, 7 C and C=O 2～3ppm.

(4) the hydrogen spectrum of ampelopsin sodium salt through being neutralized to pH6 and the hydrogen spectrum of ampelopsin, the chemical shift basically identical of each H.

The The above results explanation, after ampelopsin formed sodium salt, its A ring, 3 ring structures of B ring and C ring both did not change, and the hydrogen of prosposition still exists, and 5 hydroxyl protons and 4 oxygen have formed intramolecular hydrogen bond.Comprehensive above-mentioned data infer that each H on 3,7,3 ', 5 ' OH is replaced by Na respectively.

The molecular formula of ampelopsin sodium salt (Amp-Na) is C ₁₅H ₈O ₈Na ₄5H ₂O, relative molecular mass 498.03, its structural formula is as follows:

The preparation example II of embodiment 6 ampelopsin sodium salts

Repeat the step of embodiment 1, difference is with mol ratio to be 1: 2 Na ₂HPO ₄Replace sodium bicarbonate.

As a result, make the AMP sodium salt equally, molecular formula is: C ₁₅H ₈O ₈Na ₄5H ₂O.

Test result shows that the solvability and the stability of the ampelopsin sodium salt of formation are significantly improved equally.

The preparation example III of embodiment 7 ampelopsin sodium salts

Repeat the step of embodiment 1, difference is with mol ratio to be 1: 4 NaAc replacement sodium bicarbonate.

As a result, make the AMP sodium salt equally, molecular formula is: C ₁₅H ₈O ₈Na ₄5H ₂O.

Test result shows that the solvability and the stability of the ampelopsin sodium salt of formation are significantly improved equally.

Embodiment 8 contains the pharmaceutical composition of ampelopsin sodium salt

The pharmaceutical composition of the following prescription of preparation.

The AMP sodium salt, molecular formula is: C 15H 8O 8Na 4·5H 2O	500 milligrams
		50mM PBS damping fluid (pH 6.8)	50 milliliters

This pharmaceutical composition can be used for suppressing tumour.

Embodiment 9 contains the compound medicament composition of ampelopsin sodium salt

The pharmaceutical composition of the following prescription of preparation.

AMP sodium salt, molecular formula are C 15H 8O 8Na 4·5H 2O	500 milligrams
		?CTX?	25 milligrams
50mM PBS damping fluid (pH 6.8)	50 milliliters

This pharmaceutical composition can utilize the synergy of AMP sodium salt and CTX, reduces the side effect of CTX.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (9)

1. ampelopsin salt or derivatives thereof, wherein said derivative is a hydrate, it is characterized in that, described ampelopsin salt is to substitute the formed salt of hydrogen atom on the ampelopsin with monovalent cation,

And described ampelopsin salt has shown in the formula I:

C ₁₅H ₆O ₈H _αM _β (I)

In the formula,

M is the monovalent cation that is selected from down group: K ⁺, Na ⁺Or its combination;

α+β＝6，2≤β≤5。

2. ampelopsin salt or derivatives thereof as claimed in claim 1 is characterized in that M is Na.

3. ampelopsin salt or derivatives thereof as claimed in claim 2 is characterized in that, the derivative of described ampelopsin salt is the dihydrate or the pentahydrate of porcelain vine element unsaturated sodium salt.

4. ampelopsin salt or derivatives thereof as claimed in claim 3 is characterized in that described pentahydrate has the following formula structure:

5. method for preparing the described ampelopsin salt of claim 1 or derivatives thereof, wherein said derivative is a hydrate, it is characterized in that, comprises step:

(a) with the reaction of the salt forming agent shown in ampelopsin and the formula II, form the ampelopsin salt shown in the formula I;

Ampelopsin+M _mZ → C ₁₅H ₆O ₈H _αM _β

Formula II formula I

In the formula,

M is the monovalent cation that is selected from down group: K ⁺, Na ⁺Or its combination,

Z is the negatively charged ion that is selected from down group: HCO ₃ ^-, CO ₃ ^2-, PO ₄ ^3-, HPO ₄ ^2-, H ₂PO ₄ ^-, Ac ^-Or its combination;

M is 1,2 or 3,

α+β＝6，2≤β≤5；

Wherein in the step (a), the ratio of the mole number of M is 1: 2 to 1: 5 in ampelopsin mole number and the salt forming agent;

(b) isolate ampelopsin salt or its hydrate of formation.

6. method as claimed in claim 5 is characterized in that, described salt forming agent is selected from down group: sodium bicarbonate, yellow soda ash or its combination.

7. a pharmaceutical composition is characterized in that, it contains arbitrary described ampelopsin salt or derivatives thereof among the claim 1-4, and wherein said derivative is a hydrate.

8. pharmaceutical composition as claimed in claim 7 is characterized in that described pharmaceutical composition also contains extra antitumor drug, and wherein said antitumor drug is selected from: carboplatin, 5FU or CTX.

9. the method for a pharmaceutical compositions is characterized in that, it comprises step:

(a) the ampelopsin salt or derivatives thereof shown in the formula I is mixed with pharmaceutically acceptable carrier, wherein said derivative is a hydrate, thereby forms pharmaceutical composition:

C ₁₅H ₆O ₈H _αM _β (I)

In the formula,

M is the monovalent cation that is selected from down group: K ⁺, Na ⁺Or its combination;

α+β＝6，2≤β≤5。

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